Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. In recent years researchers have built on studies from the early 1900s to show that in many environments bacteria are found largely in biofilms and not as planktonic cells. Biofilms negatively impacts human health, in particular the formation of antibiotic resistant biofilms on a broad range of medical implant such as catheters, orthopedic implants and contact lenses is a significant clinical problem. These device-related infections cost the healthcare industry hundreds of millions of dollars annually in extended hospital stays, additional therapy and associated complications. Furthermore, biofilms can form in the absence of implants. Recent data indicate that biofilms of Pseudomonas aeruginosa form in the Cystic Fibrosis (CF) lung, likely explaining why these infections do not respond to conventional antibiotic therapy. Therefore, a better understanding of how these microbial communities form has important implications for human health. In this proposal we analyze critical set of proteins that regulate biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa, via controlling the production and degradation of the nucleotide signal cdiGMP. The studies proposed here will provide insight into mechanisms by which the cell modulates cdiGMP levels to regulate biofilm formation. Our central hypothesis is that P. aeruginosa regulates biofilm formation by modulation of cdiGMP levels, which in turn control the production of extracellular polysaccharide (EPS) and flagellar function. In this application, we focus our efforts on early events in biofilm formation under one particular set of growth conditions to explore the mechanistic underpinnings of the control of biofilm formation. We anticipate that the insight gained by these studies may be relevant to our understanding of early biofilm formation in different organisms and under a broad range of environmental conditions. A more complete understanding of the regulation of biofilm formation by this important opportunistic pathogen, and an understanding of cdiGMP signaling more generally, may provide insights into how to prevent and treat infections caused by a broad range of biofilm-forming microbes. The Specific Aims of this proposal are to: Specific Aim 1. Test the hypothesis that a second cdiGMP diguanylate cyclase (DGC) contributes to the regulation of biofilm formation. Specific Aim 2. Test the hypothesis that early biofilm formation is regulated by differential control of EPS production and flagellar function via modulating cdiGMP levels. Specific Aim 3. Test the hypothesis that pseudopilins contribute to the regulation of early biofilm formation. PUBLIC HEALTH RELEVANCE: Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. The formation of biofilms negatively impacts human health, in particular the formation of antibiotic resistant biofilms on a broad range of medical implants such as catheters and contact lenses. Device related infections cost the healthcare industry hundreds of millions of dollars annually in extended hospital stays, therapy and associated complications. Recent studies also suggest that biofilm formation plays a role in non-implant infections, for example, in addition to well-documented studies of plaque, there is emerging evidence that biofilms on host tissues in Cystic Fibrosis, otitis media (e.g., ear ache) and endocarditis. We have identified a regulatory network that regulates biofilm formation in the important opportunistic pathogen P. aeruginosa. A better understanding of this network may provide new targets for blocking the formation of these communities on medical implants and on host tissues.